| Gastric cancer is one of the most common malignant tumors in the world.According to the latest data released by the national cancer center of China in February 2018,the incidence of gastric cancer in China ranks the second among malignant tumors,with the second among men and the fifth among women.The mortality rate of malignant tumors ranks the third in China,with the third in men and the second in women.More than 80% of patients with gastric cancer were diagnosed with advanced stage of disease,and the prognosis was poor.At present,the basic diagnostic methods of gastric cancer include endoscope,biopsy specimen pathological examination and imaging examination,which are respectively used for qualitative diagnosis,localization diagnosis and staging diagnosis before treatment(c TNM).Pathological examination of mucosal biopsy during endoscope is the basis for the diagnosis and treatment of gastric cancer,and enhanced CT examination of chest,abdomen and pelvic cavity is the basic means of pre-treatment staging.In addition,chromoendoscopy,magnification endoscopy,endoscopic ultrasonography,abdominal magnetic resonance imaging(MRI),single-photon emission computed tomography(SPECT)and positron emission tomography(PET)are also used in the diagnosis of gastric cancer and the evaluation of distant metastasis.Existing endoscopy or imaging technology has its unique sensitivity and spatial resolution,but there are still some missed diagnosis rate and misdiagnosis rate.Current endoscopy and imaging examination are still based on pathological morphology changes.It is well known that molecular biology of tumor cells and tissues has often changed before the event of morphological change,so looking for new gastric cancer markers,create new endoscopic examination technology,and exploring the interdisciplinary will be good for the early diagnosis and early treatment of gastric cancer.In recent years,molecular imaging,as a new discipline,has opened up a new perspective for tumor diagnosis,which can detect the changes of diseased tissues at the cellular or even molecular level in real time and achieve the effect of "in vivo immunohistochemistry".Its emergence has brought a new revolution for the diagnosis and treatment of tumor.The clinical measurement of tumor-related biomarkers plays an important role in the diagnosis,treatment and management of tumors.Among many potential biomarkers,enzymes have received increasing attention due to their important roles in physiological,pathological and pharmacological processes.In recent years,matrix metalloproteinases(MMPs)have attracted more and more scholars’ attention.MMPs is a family of 24 zinc-dependent endopeptidases that degrade components of the extracellular matrix.Recent studies have found that MMPs expression is up-regulated in a variety of human malignant solid tumors,especially MMP-2.A meta-analysis involving 10 clinical studies found that high MMP-2 expression was significantly correlated with TNM staging,depth of infiltration,lymph node metastasis,distant metastasis,and median survival in patients with gastric cancer,suggesting that high MMP-2 expression may be a poor indicator in gastric cancer.Existing MMP-2 assays,such as immunohistochemistry,antibody labeling,and proteomics,can be used to detect and track enzymes in fixed cells or in vitro,but cannot provide real-time information on enzymes in living cells,limiting their clinical application.Because of its advantages of high sensitivity,non-invasive,rapid and real-time detection in vivo,molecule fluorescence probe imaging technology has been widely applied to the visualization and quantitative study of various dynamic processes in vivo.The fluorescence properties of the probe can be regulated by its molecular structure,which results in significant changes in the spectral characteristics of the designed probe under the interaction with some enzyme,showing high signal strength ratio and high sensitivity.Therefore,molecule fluorescent probes have become a powerful tool for real-time detection of the location and content of specific enzymes in vivo and the formation of visual fluorescence images,with great potential in the early diagnosis and treatment of cancer.In this study,we first synthesized the MMP-2 specific molecule fluorescent probe Dab-GPLG VRGY-FITC,and tested its sensitivity,specificity and cytotoxicity.Then,the probe was used to detect the expression of MMP-2 in fresh gastric cancer tissues.The probe was then incubated with the human gastric cancer cell(MGC-803)and the fluorescence imaging of MMP-2 in the cells was observed under a confocal fluorescence microscope.To evaluate whether the probe can be used for fluorescence imaging of live tumors,we further injected the probe into the tumor-bearing nude mice with gastric cancer through the caudal vein,and observed its fluorescence imaging in the small animal in vivo imaging system(IVIS).This study has completed the synthesis of molecular fluorescence probes,the imaging of clinical specimens of gastric cancer,MGC-803 gastric cancer cells and tumor-bearing animal models of gastric cancer,providing preliminary experimental basis for the further transformation of molecular fluorescence probes into clinical applications.Part Ι Synthesis of MMP-2 special fluorescent probe and assessment of its sensitivity,specificity and cytotoxicityObjective To design and synthesize a fluorescent probe which is specifically responsive to activated MMP-2,and test its sensitivity,specificity and cytotoxicity.Method The molecule fluorescent probe Dab-GPLGVRGY-FITC was prepared by the "click" chemical reaction of GPL-GVRGY-Pra catalyzed by Cu(I)and FITC-N3 and Dabcyl-NHS.The probe was reacted with MMP-2,MMP-2 inhibitor and common active enzymes(Furin,DNase,RNase and BSA),and the fluorescence intensity was detected by fluorescence spectrometer to determine the specificity of the probe to MMP-2.The probe was reacted with MMP-2 at different concentrations and its fluorescence intensity was detected by fluorescence spectrometer to determine its sensitivity to detect MMP-2.The cytotoxicity of the probe to MGC-803 cells was determined by MTT assay.Result The purity of the molecular probe was above 95%.Probe Dab-GPLGVRGY-FITC itself fluorescence intensity is weak,and the fluorescence intensity after reaction with MMP-2 significantly enhanced(40 times higher than itself),the enhancement could be effectively blocked by inhibitor of MMP-2,suggesting that the current probe possesses an excellent specificity.The fluorescence intensities of the assays increased linearly with the increasing concentrations of MMP-2,according to which a detection limit down to 14 ng/ml.The fluorescence intensity of the solution after the probe reacted with other biological enzymes(Furin,DNase,RNase and BSA)was weak.MTT cytotoxicity assay showed that when the probe concentration was within 1μM,the survival rate of MGC-803 cells after incubation with the probe was almost unchanged.When the probe concentration was increased to 10μM,the survival rate was about 85%;when the probe concentration was increased to 100μM,the survival rate was maintained at about 70%.Conclusion Probe Dab-GPLGVRGY-FITC is specifically responsive to activated MMP-2 with high sensitivity and low cytotoxicity.Part Ⅱ Imaging in clinical gastric cancer tissues in vitro with application of Probe Dab-GPLGVRGY-FITCObjective To evaluate whether the probe Dab-GPLGVRGY-FITC synthesized in part Ι could recognize clinical gastric cancer tissues in vitro and form fluorescence images.Method 11 fresh gastric cancer tissues and paracancer tissues and 1 gastritis mucosal tissue were treated with this probe.Fluorescence intensity of tissue samples and tissue lysates was detected by fluorescence spectrometer,and then the fluorescence intensity of frozen tissue sections was observed by confocal fluorescence microscopy.Result The fluorescence intensity of gastric cancer tissue reacted with probe Dab-GPLGVRGY-FITC was significantly higher than that of paracancer tissues and gastritis tissues.However,the fluorescence signal of gastric cancer and paracancer tissue pretreated with MMP-2 inhibitor significantly decreased after the reaction with the probe.The fluorescence absorbance of gastric cancer tissue lysate was 5.8-9.0 times higher than that of gastritis tissue lysate,while the fluorescence absorbance of paracancer tissue lysate was 1.0-3.1 times higher than that of gastritis tissue lysate.According to the linear relationship between MMP-2 dose and fluorescence absorbance in the first part,the content of active MMP-2 in gastric cancer tissues was estimated to be 1.26ng/mg,while the content of active MMP-2 in paracancer tissues was 0.228ng/mg.After the reaction of gastric cancer tissues with the probe,the frozen sections of gastric cancer tissues showed obvious green fluorescence under the fluorescence confocal microscope,while the fluorescence signal of paracancer tissues and gastritis tissues was weak.When the activity of MMP-2 in tissues was inhibited,the fluorescence signal of gastric cancer and paracancer tissues was significantly weakened.Conclusion The probe Dab-GPLGVRGY-FITC can be specifically combined with mmp-2 in the samples of gastric cancer tissues in vitro to form fluorescence images,and the fluorescence intensity can be used to identify gastric cancer tissues,paracancer tissues and gastritis tissues.Part Ⅲ Imaging in human gastric cancer cell MGC-803 with application of probe Dab-GPLGVRGY-FITCObjective To evaluate whether the probe Dab-GPLGVRGY-FITC synthesized in part I can distinguish gastric cancer cells from normal cells and form fluorescent images.Method The probe with concentrations of 2μM、4μM and 8μM was incubated with MGC-803 cells,and the fluorescence imaging of the cells was observed under a confocal fluorescence microscope.The probe with a concentration of 4μM was incubated with human gastric mucosal epithelial cells(GES-1)as the control group.The probe with the same concentration and the probe distribution pre-added with MMP-2 inhibitor were incubated with MGC-803 cells,and the fluorescence imaging was observed under the fluorescence confocal microscope.Result Obvious fluorescence signals could be observed in MGC-803 cells after incubation with the probe with a concentration of 2μM,and the fluorescence signals gradually increased with the increase of probe concentration.GES-1 cells showed no obvious fluorescence signal after incubation with probe with a concentration of 4μM,while MGC-803 cells showed obvious fluorescence signal after incubation with the probe with the same concentration,while MGC-803 cells pretreated with MMP-2 inhibitor showed significantly weakened fluorescence signal.Conclusion The probe Dab-GPLGVRGY-FITC can specifically bind to MMP-2 in human gastric cancer cell MGC-803 and form fluorescence images.Part Ⅳ In vivo imaging in tumor-bearing nude mice with gastric cancer with application of probe Dab-GPLGVRGY-FITCObjective To evaluate whether the probe Dab-GPLGVRGY-FITC synthesized in part I could image tumor tissues of nude mice with gastric cancer by identifying MMP-2 and preliminarily evaluate its toxicity.Method The probe was injected into the tumor-bearing nude mice via caudal vein and imaged in the in vivo imaging system(IVIS).To reduce the self-fluorescence in nude mice,tumor tissues were further removed and observed in IVIS.Blood of nude mice was collected for blood routine and biochemical tests,and compared with the control group.Result Fluorescence signals appeared at the tumor site in the IVIS system as early as 15min after the probe was injected into the tumor-bearing nude mice,and the fluorescence signals in the tumor area gradually increased with the passage of time.In vitro tumor tissues,fluorescence signal was also significantly enhanced in IVIS,which could be blocked by MMP-2 inhibitor.Compared with the control group,red blood cell count(RBC)and platelet count(PLT)of the probe group decreased significantly(P <0.05),which were(4.88±0.05 vs 5.20±0.04)×1012/L and(612±8.60 vs 657.5±4.95)×1012/L,respectively.There was no significant difference in white blood cell count(WBC)and hemoglobin(HB)between the two groups(P >0.05).The AST and ALT levels in the probe group were significantly higher compared with the control group(P <0.05),which were(49.66±3.39 vs 34.75±0.64)U/L and(32.27±2.94 vs 17.18±0.21)U/L,respectively.There was no significant difference in alkaline phosphatase(ALP),Gamma Glutamyl Transpeptidase(GGT)and total bilirubin(TB)between the two groups(P >0.05).Serum creatinine(Scr)in the probe group was higher than that in normal nude mice(P <0.05),which was(55.72±1.80 vs 54.10±0.50)mmol/L.There was no significant difference in blood glucose(GLU)and total cholesterol(TC)between the two groups(P >0.05).Conclusion The probe Dab-GPLGVRGY-FITC can form fluorescent images in tumor tissues of tumor-bearing nude mice by specifically binding the MMP-2,and the probe has low hepatorenal toxicity. |
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